The long-term goal of the proposed research is to utilize novel allosteric modulators of the M5 muscarinic receptor (mAChR) to elucidate the physiological consequence of M5 receptor activation in midbrain dopaminergic neurons. Dysregulation of dopamine release in the nucleus accumbens (NAc) and striatum is thought to underlie multiple central nervous system (CNS) disorders including Parkinson's disease, attention deficit disorder (ADHD), and drug addiction. Stimulation of cholinergic afferents to dopaminergic neurons in the substantia nigra pars compacta (SNc) and ventral tegemental area (VTA) can increase dopamine release in the NAc and striatum respectively, an effect that is sensitive to mAChR antagonists. The only detectable mAChR on these midbrain dopaminergic neurons is of the M5 variety and studies in M5 knock- out mice support the hypothesis that M5 activation leads to excitation of these dopaminergic neurons. However, validation of the therapeutic potential of M5 has awaited the development of an M5-selective modulator. The development of selective orthosteric agonists for the M5 receptor has been relatively unsuccessful due to the high conservation of the orthosteric, acetylcholine (ACh) binding site across all five muscarinic receptor subtypes (M1-M5). However, by targeting allosteric sites that are removed from the orthosteric pocket the Conn lab has successfully developed both a positive and negative allosteric modulator of the M5 receptor (VU0238429 and VU0419959 respectively). In these studies I propose to use molecular pharmacological techniques to fully characterize the mechanism whereby these M5-selective modulators alter ACh-mediated signaling. I then plan to perform electrophysiology studies in brain slices to test the hypothesis that carbachol-induced inward currents in dopamine neurons are mediated by M5 receptor activation. Finally, I aim to use to use microdialysis to determine if infusion of these modulators into the SNc and VTA can alter extracellular dopamine levels in awake, freely moving rats. I hypothesize that the M5 positive modulator will increase dopamine neuron inward currents and increase dopamine release while the negative modulator will decrease both excitability and extracellular dopamine levels. These studies are important to both elucidate the physiological role of M5 as well as to validate the therapeutic potential of M5 modulation in multiple CNS diseases such as Parkinson's Disease, ADHD, and drug addiction. PUBLIC HEALTH RELEVANCE: Alterations in midbrain dopaminergic neuron activity have been implicated in several central nervous systems disorders including Parkinson's disease, attention deficit disorder, and addiction. Eludicating the physiological role of M5 and other muscarinic receptor subtypes in regulating dopaminergic neurons via subtype-specific allosteric modulators will have important implications for the development of novel approaches to treat these diseases.